ABSTRACT Noise can irreversibly damage the sensory receptors of the inner ear, the hair cells, and lead to permanent hearing loss. Hair cells respond to sound-induced mechanical vibrations with inward current, passing through mechanotransduction channels at the tips of stereocilia. In response to stereocilia deflections, calcium ions enter the cell, bind to fixed and mobile buffers and are extruded by cell membrane calcium pumps. The basal, high frequency outer hair cells have relatively few calcium pumps. Thus, mitochondria, which are concentrated in a belt beneath the cuticular plate, a support structure for stereocilia, may play a major role in calcium removal during mechanical stimulation in these cells. The goal of this study is to understand how mechanical overstimulation impairs mechanotransduction, perturbs calcium balance, and disrupts redox homeostasis in cochlear hair cells. This will aid in understanding why basal, high frequency outer hair cells are more susceptible to noise exposure than apical, low frequency ones. In addition we will test a novel strategy to increase hair cell viability by protecting their mitochondria from calcium overload and oxidative damage. The first aim of our proposal is to determine changes in hair cell mechanotransduction function following mechanical overstimulation. Our preliminary data show significant calcium increases in the basal, high frequency outer hair cells during mechanical stimulation. Calcium was promptly removed from the cytosol following stimulation, while mitochondrial calcium overload was sustained. Because an increase in mitochondrial calcium is commonly reported in cell death pathways, a study of hair cells' mitochondrial calcium balance following overstimulation is necessary. Within the second aim, we will use a knockout mouse model with impaired mitochondrial calcium uptake to determine whether mitochondrial calcium overload is required for oxidative stress in hair cells following overstimulation. When overloaded with Ca, mitochondriaundergo oxidative damage; this will further increase oxidative stress, initiating a feed-forward cycle to further damage mitochondria. We will test the hypothesis that mitochondrial calcium overload and dysfunction in outer hair cells contribute to the vulnerability of these sensory cells to overstimulation. To complement our genetic studies, we will also examine whether protecting mitochondria against calcium overload and oxidative stress will increase hair cell viability by using a pharmacological approach: we will use novel Szeto-Schiller peptides that concentrate to the inner mitochondrial membrane to protect mitochondria from oxidative damage and improve mitochondrial bioenergetics. In summary, our study will reveal how mechanical overstimulation can damage the stereociliary bundle and impair its function. In addition, this study will establish the role of mitochondrial Ca2+ overload in hair cell dysfunction and death, providing not only a mechanistic understanding of the process, but possibly paving the way for a novel clinical strategy to protect cochlear hair cells.